A key factor in the recent advances in molecular biology has been the use of oligonucleotide analogs to study and enable basic cellular processes, including the regulation of gene expression. One of the most powerful and versatile tools in molecular biology is the in vitro replication of nucleic acid sequences, as exemplified by the ubiquitous practice and commercial value of the polymerase chain reaction (PCR) and DNA sequencing. Both methods entail hybridization of a primer, usually a short (15-30 nt) synthetic oligonucleotide, to a single-stranded template nucleic acid. A polymerase enzyme catalyzes extension, i.e., polymerization, from the 3' terminus of the primer with 5' triphosphate nucleotides complementary to the template strand. By this general replication method, sequencing information may be generated, or amplification of the template may be achieved through the choice of selected variables such as primers, multiple primers, enzymes, nucleotides, and the selection of buffers, salts, temperature, and temperature cycling conditions.
Many internucleotide analogs of DNA have been synthesized, primarily for study of their antisense effects, the inhibition of gene expression at the transcriptional level, targeting DNA, or more commonly pre-translational targeting mRNA. The antisense effect includes occupying a critical gene expression site in a sequence-specific effect with a high-affinity, nuclease-resistant oligonucleotide analog or the RnaseH mediated cleavage of mRNA in the duplex formed with the antisense oligonucleotide. The latter effect results in destruction of genetic message. Both strategies have the intended effect of precluding formation of the undesired gene product, typically of viral origin.
Internucleotide DNA analogs with a bridging nitrogen, especially replacing the oxygen at the 3' of the deoxyribose moieties, have markedly different physical properties when compared with DNA. These DNA analogs having nitrogen replacement of oxygen at the 3' deoxyribose moiety are commonly referred to as phosphoramidates (Gryaznov et al., Nuc. Acids Res. 24:1508-1514 (1996). Phosphoramidate containing oligonucleotides have been shown to have greater affinity for their complementary DNA and RNA, exemplified by higher thermal melting values, T.sub.m. In this effect, affinity is synonymous with hybridization strength and duplex stability. Phosphoramidate oligonucleotides demonstrate a high degree of base-discrimination to pair with a complementary strand following the normal Watson-Crick rules. The level of discrimination is often termed specificity. Affinity may be measured in experiments that compare the T.sub.m values of duplexes having perfect Watson-Crick complementarity versus those with one or more mismatches. The destabilization, seen by the decrease in T.sub.m, is a measure of specificity, pertinent to structural modifications, hybridization conditions, or other experimental parameters.
Additionally, phosphoramidate oligonucleotides may form triple helical structures, involving three strands in a sequence dependent manner. Triplex structures can result from directly targeting double-stranded DNA with phosphoramidate oligonucleotides. Phosphoramidate oligonucleotides are poor substrates for phosphodiesterase, exo- and endonucleases which rapidly degrade foreign DNA in cells. Thus, such analogs may exert their antisense and other hybridization-dependent effects, over a useful period of time in vitro or in vivo.
Although many DNA analogs have some desirable properties, such analogs may have numerous other properties that render them unsuitable for common molecular biology techniques such as PCR or nucleic acid sequencing. For example, peptide nucleic acid (PNAs) cannot serve as replication template or function as synthesis primers. Similarly, DNA analogs having only phosphoramidate linkages between nucleosides have been found not to function as synthesis primers.
Accordingly, it is of interest to provide new polynucleotide analogs that have one or more properties that are advantageous with respect to corresponding DNA molecules, but may also be used in a variety of molecular biology methods including PCR and other primer extension reactions. It is also of interest to provide methods of using such analogs.